Fig. 5: CXCR3 inhibition exerts synergistic cytotoxic effects when combined with AC220, and CXCL10 stimulation partly rescues the apoptosis induced by cotreatment of AC220 and DAPT.

a MOLM13 and b MV4-11 cells were treated with AC220 (0–5 nM) either alone or in combination with AMG487 (0–25 µM) for 72 h, and cell proliferation was measured in triplicate using the CCK-8 assay. c Annexin V binding was assessed in MOLM13 and MV4-11 cells treated with AC220 (2.5 nM) and/or AMG487 (10 µM) in triplicate for 48 h. Data represent the average of three independent experiments ± SD. (*P < 0.05; **P < 0.01, ***P < 0.001). d MOLM13 and MV4-11 cells were treated with AC220 (2.5 nM) either alone or in combination with AMG487 (10 µM) for 12 h, and expression levels of CXCR3, phospho-ERK (pERK), total ERK, and GAPDH were measured by western blotting of cellular proteins. Images are representative of three independent experiments. MOLM13 and MV4-11 cells were treated with DMSO, AC220 (2.5 nM), DAPT (25 µM), or Combo (AC220 combined with DAPT), in addition to either vehicle or CXCL10 (10 ng/ml). e The expression of CXCR3 at 12 h was measured in triplicate by qPCR relative to GAPDH expression. f Apoptosis at 48 h was measured in triplicate by Annexin V staining. g Expression of Hes1 at 12 h was measured in triplicate by qPCR relative to GAPDH expression. Data represent the average of three independent experiments ±SD. (*P < 0.05, **P < 0.01, ***P < 0.001). h Primary AML samples (n = 7) were treated with AC220 (250 nM) and/or AMG487 (10 μM) for 48 h. i Apoptosis at 48 h was measured in triplicate by Annexin V staining. The corresponding clinical parameters for each AML patient are presented below. Samples were ranked by their apoptosis rate under the combined treatment. Statistical analysis of apoptosis is presented on the left. FLT3/ITD⁺ (n = 3) and FLT3/WT (n = 4). Data represent the average of three independent experiments ±SD. (*P < 0.05, **P < 0.01).